Stimuli-responsive polymer utilizing keto-enol tautomerization and stimuli-responsive separating material and chemical-releasing capsule comprising the same

ABSTRACT

A stimuli-responsive polymer derivative utilizing keto-enol tautomerization. Also disclosed are a simple process for producing an N-acyl(meth)acrylamide derivative which can be used as a monomer for the stimuli-responsive polymer, a process for the production of an intermediate thereof, and an intermediate thus produced.

FIELD OF THE INVENTION

[0001] The present invention relates to an excellent stimuli-responsivepolymer derivative which can be used for drug delivery system (DDS),chemovalve, various separating agents, catheter, artificial muscle, etc.

BACKGROUND OF THE INVENTION

[0002] In recent years, stimuli-responsive polymers have been widelyused for drug delivery system (DDS), various separating agents,catheter, artificial muscle, chemovalve, etc. and thus have been ofgrowing importance. For example, JP-A-8-103653 (The term “JP-A” as usedherein means an “unexamined published Japanese patent application”)discloses a polymer which changes in its higher order structure to swellor shrink in an aqueous solution by the action of heat, light or by achange in pH or potential as a stimuli-responsive polymer. Specifically,acrylamide or methacrylamide derivatives such aspoly-N-isopropylacrylamide, N,N-diethylacrylamide andN-isopropylmethacrylamide, and vinylethers such as vinyl methyl etherare disclosed as a polymer having an upper critical solution temperature(UCST) or a lower critical solution temperature (LCST) with respect towater, which swells or shrinks in response to a temperature change.

[0003] Although these known polymers which swell or shrink in responseto a temperature change are described as having an upper criticalsolution temperature (UCST) or a lower critical solution temperature(LCST), they all have, in fact, a lower critical solution temperature(LCST). In other words, at a temperature of not lower than the lowercritical solution temperature, these polymers reversibly undergoagglomeration of polymers that renders themselves insoluble in water. Onthe contrary, at a temperature of not higher than the lower criticalsolution temperature, these polymers can be dissolved in water. Forexample, poly-N-isopropylacrylamide (PNIPAM), which is applied to DDS,etc. at present, has a lower critical solution temperature of 32° C. inan aqueous solution. When this polymer is allowed to gel, it reversiblyundergoes swelling and shrinkage depending on the temperature developedby heat.

[0004] A polymer having a lower critical solution temperature (LSCT)shrinks at a predetermined temperature or higher and thus isdisadvantageous in that it can be hardly adjusted so as to meet thedemand for shrinkage at low temperature (preferably not higher than thebody temperature) in the application to DDS, separating agent, etc.

[0005] However, all these known thermo-responsive polymers such aspoly-N-isopropylacrylamide are stimuli-responsive polymers having alower critical solution temperature (LCST) which respond only to thermalstimulation. Thus, these thermo-responsive polymers can neither switch alower critical solution temperature to an upper critical solutiontemperature (UCST) nor have, in a single compound, both functions ofcausing their reversible dissolution and precipitation depending on thehydrogen ion concentration, when they respond to heat.

[0006] On the other hand, as the polymer which changes in its higherorder structure by a pH change there is known a polyacrylic acid orpolymethacrylic acid. However, these compounds contain carboxylic acid,which has electric charge, and thus are disadvantageous in that aseparating agent comprising such a polymer adsorbs compounds other thandesired compounds (non-specific adsorption) and thus cannot provideefficient separation and purification.

[0007] If a composite stimuli-responsive polymer which can switchbetween a lower critical solution temperature (LCST) and an uppercritical solution temperature (UCST) or have, in a single compound, bothfunctions of causing its reversible dissolution and precipitationdepending on the hydrogen ion concentration can be obtained, the abovedescribed adjustment can be easily conducted. The appearance of such apolymer has been desired particularly in an art requiring fineadjustment because such a thermo-responsive polymer can be more widelyused.

[0008] Further, if used as a separating agent for protein inert to heat,etc., the conventional polymer agglomerates when heated, causingdenaturation of protein.

[0009] Moreover, if the polymer is used as DDS (e.g., chemical-releasingcapsule) by encapsulating a chemical in its gel, it is necessary thatthe affected part be cooled to allow the gel to swell and release thechemical upon releasing. However, it is practically easy to raise,rather than cool, the temperature of the affected part.

[0010] Further, if a thermo-responsive polymer is used as DDS, it needsto exhibit an upper critical solution temperature (UCST) inphysiological saline. In this respect, an interpenetration polymernetwork (IPNa) of polyacrylic acid and polyacryloyl glycinamide is knownas a thermo-responsive polymer which exhibits an upper critical solutiontemperature (UCST) in an aqueous solution (Makromol. Chem., RapidCommun. 13, 557-581 (1992)). However, this polymer does not exhibit anyupper critical solution temperature (UCST) in physiological saline.

[0011] Therefore, the appearance of a thermo-responsive polymer whichagglomerates when heated in an aqueous solution and exhibits an uppercritical solution temperature (UCST) even in physiological saline hasbeen desired.

SUMMARY OF THE INVENTION

[0012] An object of the present invention is to provide solution to theabove described problems.

[0013] A first object of the present invention is to provide astimuli-responsive polymer which exhibits an upper critical solutiontemperature (UCST) or a stimuli-responsive polymer which undergoesreversible dissolution and precipitation depending on the hydrogen ionconcentration or an addition of a solvent.

[0014] A second object of the present invention is to provide athermo-responsive polymer which exhibits both a lower critical solutiontemperature (LCST) and an upper critical solution temperature (UCST).

[0015] A third object of the present invention is to provide a compositestimuli-responsive polymer which can switch between a lower criticalsolution temperature (LCST) and an upper critical solution temperature(UCST) or can have, in a single compound, both functions of causing itsreversible dissolution and precipitation depending on the hydrogen ionconcentration.

[0016] A fourth object of the present invention is to provide athermo-responsive polymer which agglomerates when heated in an aqueoussolution and exhibits an upper critical solution temperature (UCST) evenin physiological saline.

[0017] Further, the present invention also concerns a simple process forproducing an N-acyl(meth)acrylamide derivative which can be used as amonomer for the stimuli-responsive polymer, a process for the productionof an intermediate thereof, and an intermediate thus produced.

[0018] A first aspect of the present invention concerns the followingpolymer derivatives:

[0019] 1-1) A stimuli-responsive polymer derivative having an uppercritical solution temperature utilizing keto-enol tautomerization.

[0020] 1-2) A stimuli-responsive polymer derivative utilizing keto-enoltautomerization which undergoes phase transition by a change in hydrogenion concentration or by an addition of an organic solvent.

[0021] 1-3) The stimuli-responsive polymer derivative according to theabove 1-1) or 1-2), which comprises as a polymerizable component amonomer represented by the following general formula (1):

[0022] wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; R² represents a single bond or a C₁₋₄straight-chain or branched alkylene group which may be halogenated; R³,R⁴ and R⁵ each independently represents a hydrogen atom or methyl group;and X and X′ each independently represents an oxygen atom, sulfur atom,selenium atom or tellurium atom.

[0023] 1-4) The stimuli-responsive polymer derivative according to theabove 1-1) or 1-2), which comprises as copolymerizable components ahydrophilic or hydrophobic monomer and a monomer represented by generalformula (1).

[0024] 1-5) The stimuli-responsive polymer derivative according to theabove 1-3), which comprises as a polymerizable component a monomerrepresented by the following general formula (7):

[0025] 1-6) A stimuli-responsive separating agent comprising astimuli-responsive polymer derivative according to any one of the above1-1) to 1-5).

[0026] The inventors paid attention to strong hydrogen bondingproperties represented by peptide bond and reversible keto-enoltautomerization. On the supposition that a thermo-responsive polymerhaving an upper critical solution temperature (UCST) can be obtainedusing keto-enol switching as shown in the following reaction formula A,the present invention has been worked out.

[0027] In other words, the chemical reaction was designed using acomputerized method for the calculation of molecular orbital such thatenolation occurs at a high temperature to effect hydration andconversion to keto form occurs at a low temperature to effectagglomeration by hydrogen bond. As a result, it was found that the abovedescribed design allows the appearance of an upper critical solutiontemperature (UCST). More particularly, it is preferred to synthesize acompound in which the site having a peptide bond is thermodynamicallystable in its keto form.

[0028] Further, the above described theory gives a finding that theutilization of keto-enol tautomerization makes it possible to make theabove described keto-enol switching (reversible conversion between ketoform and enol form) effectively not only by a thermal change but also bya change in hydrogen ion concentration or an addition of an organicsolvent, i.e., to obtain a stimuli-responsive polymer which reversiblyrepeats swelling and shrinkage in accordance with a change in hydrogenion concentration or an addition of an organic solvent without raisingor lowering the temperature.

[0029] The stimuli-responsive polymer derivative of the present aspectof the present invention can be effectively applied to the separation,fixing, calibration and control of various substances. In particular,the stimuli-responsive polymer derivative exhibits an upper criticalsolution temperature (UCST), i.e., agglomerates when the temperaturelowers or reversibly repeats swelling and shrinkage in accordance with achange in hydrogen ion concentration or an addition of an organicsolvent without raising or lowering the temperature. Accordingly, it isparticularly useful for the separation, purification, fixing,calibration and control of substances which are desirably not to be in ahigh temperature atmosphere (protein such as biological product, enzymeand antibody).

[0030] A second aspect of the present invention concerns a copolymerderivative comprising a monomer component having a lower criticalsolution temperature (LCST) and a monomer component having an uppercritical solution temperature (UCST).

[0031] As the monomer component having a lower critical solutiontemperature (LCST), a monomer represented by any one of the followinggeneral formulae (2) to (5) can be used:

[0032] wherein R¹ represents a hydrogen atom or a methyl group; R² andR³ each independently represents a hydrogen atom or a C₁₋₁₀straight-chain, branched or cyclic alkyl, alkoxyl, alkylamino, aryl orheterocyclic group which may be halogenated; R⁴ represents a C₁₋₁₀straight-chain, branched or cyclic alkyl or alkylakoxyl group which maybe halogenated; R⁶ represents a C₁₋₁₀ straight-chain, branched or cyclicalkyl, alkoxyl, alkylamino, aryl or heterocyclic group which may behalogenated; and n represents an integer of 4 or 5.

[0033] As the monomer component having an upper critical solutiontemperature (UCST), a monomer represented by the above described generalformula (1) can be preferably used.

[0034] The stimuli-responsive polymer derivative of the presentinvention may further comprise as a third component a hydrophilic orhydrophobic copolymerizable monomer incorporated therein. The transitionpoint of the stimuli-responsive polymer derivative can be controlled bythe incorporation.

[0035] It can be presumed that the monomer component represented by theabove described general formula (1) exhibits strong hydrogen bondingproperties represented by peptide bond and a reversible keto-enoltautomerization and has an upper critical solution temperature (UCST)developed by keto-enol switching as shown in the following reactionformula A:

[0036] In other words, the chemical reaction was designed using acomputerized method for the calculation of molecular orbital such thatenolation occurs at a high temperature to effect hydration andconversion to keto form occurs at a low temperature to effectagglomeration by hydrogen bond. As a result, it was found that the abovedescribed design allows the appearance of an upper critical solutiontemperature (UCST). More particularly, it is preferred to synthesize acompound in which the site having a peptide bond is thermodynamicallystable in its keto form.

[0037] The thermo-responsive copolymer derivative of the present aspectof the present invention can be effectively applied to the separation,fixing, calibration and control of various substances. In particular,the thermo-responsive copolymer derivative of the present aspect of thepresent invention has both an upper critical solution temperature (UCST)and a lower critical solution temperature (LCST). Accordingly, it can beeffectively used for the separation, purification, fixing, calibrationor control of substances the working temperature of which can be hardlypredetermined (protein such as biological product, enzyme and antibody).Alternatively, it can be effectively used for chemovalve.

[0038] A third aspect of the present invention concerns a compositestimuli-responsive polymer derivative having a lower critical solutiontemperature (LCST) comprising at least one monomer component representedby the following general formula (6):

[0039] wherein R⁵ represents a hydrogen atom or a methyl group; and R¹represents a hydrogen atom or a C₁₋₁₀ straight-chain, branched or cyclicalkyl, alkoxyl, alkylamino, aryl or heterocyclic group which may behalogenated. The compounds of general formula (6) corresponds tocompounds of general formula (1) wherein R² represents a single bond, R³and R⁴ each represents a hydrogen atom, and X and X′ each represents anoxygen atom.

[0040] The above described polymer derivative acts as athermo-responsive polymer which exhibits a lower critical solutiontemperature (LCST) in an aqueous solution. The lower critical solutiontemperature (LCST) can be reversibly changed by the hydrogen ionconcentration. In other words, the above described polymer derivativeshows a composite stimulation response, that is, individually respondsto heat and pH when stimulated by pH.

[0041] Further, when put in an aqueous solution having a small amount ofan organic solvent added thereto, this thermo-responsive polymer losesthe lower critical solution temperature (LCST), which has appeared sofar, but exhibits an upper critical solution temperature (UCST). Inother words, when stimulated by an organic solvent added, thisthermo-responsive polymer undergoes conversion of lower criticalsolution temperature to upper critical solution temperature.

[0042] It is particularly preferred that the stimuli-responsive polymerderivative of the present aspect of the present invention be a copolymerderivative comprising as a copolymerizable component at least onemonomer component which is hydrophilic or hydrophobic with respect tothe monomer component represented by general formula (6).

[0043] The term “monomer component which is hydrophilic or hydrophobicwith respect to the monomer component represented by general formula(6)” as used herein is intended to mean, if the monomer of generalformula (6) is hydrophobic, a monomer component which is morehydrophilic than the hydrophobic monomer component of general formula(6), and if the monomer of general formula (6) is hydrophilic, a monomercomponent which is more hydrophobic than the hydrophilic monomercomponent represented by general formula (6). The hydrophilic orhydrophobic monomer may be a monomer component represented by generalformula (6) so far as it is hydrophilic or hydrophobic with respect tothe one monomer component represented by general formula (6). In thiscase, the hydrophilic or hydrophobic monomer contains two or moremonomer components represented by general formula (6).

[0044] Accordingly, a preferred embodiment of the present aspect of thepresent invention is a copolymer further comprising at least one monomercomponent which is hydrophilic or hydrophobic with respect to onemonomer component represented by general formula (6) (including amonomer component represented by general formula (6)).

[0045] The content of the above described hydrophilic or hydrophobicmonomer is preferably from 1 to 70% by weight, more preferably from 3 to50% by weight based on the total weight of the polymer. When the contentof the above described hydrophilic or hydrophobic monomer falls withinthe above defined range, the above described properties of the presentaspect of the present invention can be exerted particularly effectively.

[0046] The thermo-responsive polymer derivative of the present aspect ofthe present invention can be effectively applied to the separation,fixing, calibration or control of various substances. In particular, thethermo-responsive polymer derivative of the present aspect of thepresent invention is a composite stimuli-responsive polymer which hasboth an upper critical solution temperature (UCST) and a lower criticalsolution temperature (LCST) within various temperature ranges andresponds also to hydrogen ion concentration. Accordingly, it can beeffectively used for the separation, purification, fixing, calibrationor control of substances the working temperature of which can be hardlypredetermined (protein such as biological product, enzyme and antibody)Alternatively, it can be effectively used for chemovalve.

[0047] A fourth aspect of the present invention concerns athermo-responsive polymer derivative having an upper critical solutiontemperature (UCST) in an aqueous solution, which comprises at least onemonomer component represented by the following general formula (6) andat least one monomer component selected from acrylamide andmethacrylamide:

[0048] wherein R⁵ represents a hydrogen atom or methyl group; and R¹represents a hydrogen atom or a C₁₋₁₀ straight-chain, branched or cyclicalkyl, alkoxyl, alkylamino, aryl or heterocyclic group which may behalogenated.

[0049] The thermo-responsive polymer derivative of the present aspect ofthe present invention is a copolymer derivative comprising at least onemonomer component represented by general formula (6) and at least onemonomer component selected from acrylamide and methacrylamide ascopolymerizable components.

[0050] In the present aspect of the present invention, the chargedproportion of the monomer represented by general formula (6) ispreferably from 0.1 to 100% by weight, more preferably from 1 to 30% byweight, particularly from 5 to 15% by weight based on the weight ofacrylamide and/or methacrylamide.

[0051] The thermo-responsive polymer derivative of the present aspect ofthe present invention may further comprise at least one hydrophilic orhydrophobic monomer component which is hydrophilic or hydrophobic withrespect to monomer component represented by general formula (6)(excluding acrylamide and methacrylamide) incorporated therein as acopolymerizable component as necessary. The term “monomer componentwhich is hydrophilic or hydrophobic with respect to the monomercomponent represented by general formula (6)” as used herein is intendedto mean, if the monomer of general formula (6) is hydrophobic, a monomercomponent which is more hydrophilic than the hydrophobic monomercomponent of general formula (6), and if the monomer of general formula(6) is hydrophilic, a monomer component which is more hydrophobic thanthe hydrophilic monomer component represented by general formula (6).The hydrophilic or hydrophobic monomer may be a monomer componentrepresented by general formula (6) so far as it is hydrophilic orhydrophobic with respect to the one monomer component represented bygeneral formula (6). In this case, the hydrophilic or hydrophobicmonomer contains two or more monomer components represented by generalformula (6).

[0052] The charged proportion of the above described hydrophilic orhydrophobic monomer is preferably from 1 to 70% by weight, morepreferably from 3 to 50% by weight based on the total weight of themonomer component represented by general formula (6) and acrylamideand/or methacrylamide.

[0053] The thermo-responsive polymer derivative of the present inventionexhibits an upper critical solution temperature (UCST) in an aqueoussolution, particularly physiological saline, and thus can be effectivelyapplied to the separation, fixing, calibration or control of varioussubstances. Accordingly, it can be effectively used for the separation,purification, fixing, calibration or control of substances the workingtemperature of which can be hardly predetermined (protein such asbiological product, enzyme and antibody). Alternatively, it can beeffectively used for chemovalve, drug delivery system (DSS), etc.

DETAILED DESCRIPTION OF THE INVENTION

[0054] The present invention will be further described hereinafter.

[0055] In accordance with the first aspect of the present invention, asmentioned above, by properly making a molecular design using keto-enoltautomerization, a stimuli-responsive polymer derivative having an uppercritical solution temperature (UCST) or a stimuli-responsive polymerderivative which undergoes phase transition in accordance with a changein hydrogen ion concentration or an addition of an organic solvent canbe easily obtained.

[0056] For example, a polymer derivative containing a substituentcomponent represented by the following general formula (8) may bepreferably used:

[0057] wherein R¹, X and X′ are the same as those defined in generalformula (1), respectively. The preferred ranges thereof are also thesame as those described below with reference to general formula (1).

[0058] In the polymer derivative containing a substituent componentrepresented by general formula (8) (hereinafter described with referenceto the case where X and X′ each represents an oxygen atom for thesimplification of description), the amide bonding site shows reversibleswitch between keto form and enol form as shown in the followingreaction formula B in accordance with an application of heat, a changein the hydrogen ion concentration, or with an addition of an organicsolvent.

[0059] Further, the present inventors found that a polymer derivativecontaining a monomer represented by the above described general formula(1) as a polymer component is particularly effective for efficientreversible keto-enol conversion.

[0060] In general, a compound having an amide bond itself agglomeratesdue to strong hydrogen bonding in an aqueous solution. A polyamide whichtakes a keto form in an aqueous solution is insoluble in water. However,it can be presumed that this keto form is converted to an enol form dueto heat or a change in hydrogen ion concentration to lose itsself-agglomeration effect, giving a water-soluble compound.

[0061] The monomer represented by general formula (1) is described inmore detail below.

[0062] In general formula (1), R¹ preferably represents a C₁₋₈straight-chain, branched or cyclic alkyl, alkoxyl, alkylamino or phenylgroup, more preferably methyl, ethyl, propyl, isopropyl, phenyl,methoxy, propoxyl, isopropoxyl, methylamino or ethylamino group,particularly methyl, ethoxy or methylamino group. These groups may besubstituted by a halogen atom such as fluorine, bromine, chlorine andiodine. Particularly preferred substituents are fluorine atom andchlorine atom.

[0063] R² preferably represents a single bond or C₁₋₂ straight-chain orbranched alkylene group or halogenated alkylene group, particularly asingle bond. Preferred examples of substituents on the alkylene groupinclude fluorine atom and chlorine atom.

[0064] X and X′ each is preferably an oxygen atom or sulfur atom.

[0065] Examples of the monomer represented by general formula (1)include N-acetylacrylamide, N-fluoroacetyl acrylamide,N-propionylacrylamide, N-butanoylacrylamide, N-pentanoylacrylamide,N-hexanoylacrylamide, N-isobutanoyl acrylamide, N-benzoylacrylamide,N-(3-fluorobenzoyl) acrylamide, N-(2, 3-difluorobenzoyl)acrylamide,N-pyridylcarbonylacrylamide, N-pyrimidylcarbonylacrylamide,N-acetylmethacrylamide, N-fluoroacetylmethacrylamide,N-propionylmethacrylamide, N-butanoylmethacrylamide,N-pentanoylmethacrylamide, N-hexanoylmethacrylamide,N-isobutanoylmethacrylamide, N-benzoylmethacrylamide,N-(3-fluorobenzoyl)methacrylamide, N-(2, 3-difluorobenzoyl)methacrylamide, N-pyridylcarbonylmethacrylamide, N-pyrimidylcarbonylmethacrylamide, N-acroyl-N′-methylurea, N-acroyl-N′-ethylurea,N-acroyl-N′-fluoromethylurea, N-acroyl-N′-difluoromethylurea,N-acroyl-N′-trifluoromethylurea, N-methacroyl-N′-methylurea,N-methacroyl-N′-ethylurea, N-methacroyl-N′-fluoromethylurea,N-methacroyl-N′-difluoromethylurea, N-methacroyl-N′-trifluoromethylurea,methyl N-acroylcarbamate, ethyl N-acroylcarbamate, n-propylN-acroylcarbamate, isopropyl N-acroylcarbamate, n-butylN-acroylcarbamate, isobutyl N-acroylcarbamate, fluoromethylN-acroylcarbamate, difluoromethyl N-acroylcarbamate, trifluoromethylN-acroylcarbamate, 2, 2, 2-trifluoroethyl N-acroylcarbamate, methylN-methacroyl carbamate, ethyl N-methacroylcarbamate, n-propylN-methacroyl carbamate, isopropyl N-methacroylcarbamate, n-butylN-methacroyl carbamate, isobutyl N-methacroylcarbamate, t-butylN-methacroylcarbamate, fluoromethyl N-methacroylcarbamate,difluoromethyl N-methacroylcarbamate, trifluoromethylN-methacroylcarbamate, and 2, 2, 2-trifluoroethyl N-methacroylcarbamate.

[0066] Specifically, homopolymerization of a monomer represented bygeneral formula (1) or copolymerization of a monomer represented bygeneral formula (1) with a hydrophilic or hydrophobic monomer makes itpossible to obtain thermo-responsive polymers having a UCST withinvarious temperature ranges, pH-responsive polymers which respond tovarious hydrogen ion concentrations or solvent-responsive polymers whichrespond to an addition of an organic solvent.

[0067] Further, copolymerization of a monomer represented by generalformula (1) with a monomer for a thermo-responsive polymer having anLCST makes it possible to obtain a heat-and pH-responsive polymer whichexhibits both heat response and pH response.

[0068] The thermo-responsive polymer having a UCST preferably exhibitsan upper critical solution temperature of from 0 to 50° C., particularlyfrom 0 to 38° C., if it is used as a separating agent.

[0069] Further, the switching range of the thermo-responsive polymer(range of phase transition temperature) is preferably as narrow aspossible. In accordance with the present invention, a thermo-responsivepolymer having a practical switching range of higher than 10° C. can beobtained.

[0070] The organic solvent to be used for stimulation is notspecifically limited so far as it has some solubility in water. Specificexamples of the organic solvent include methanol, ethanol, propanol,isopropanol, acetone, THF, dioxane, acetic acid, propionic acid,ethylene glycol, and propylene glycol. Preferred among these organicsolvents are methanol, ethanol, propanol, isopropanol, acetone, and THF.These organic solvents can efficiently accelerate the agglomeration ofthe stimuli-responsive polymer, though depending on the kind of thestimuli-responsive polymer.

[0071] It was also found that the application of stimulation by anorganic solvent makes it possible to develop a keto-enol switching typeheat response.

[0072] Further, the novel stimuli-responsive polymer derivative of thepresent invention is effective for the separation, fixing, calibrationor control of substances which are desirably not to be in a hightemperature atmosphere. It can be effectively applied to drug deliverysystem (DDS), various separating agents, catheter, artificial muscle,etc.

[0073] In particular, the stimuli-responsive polymer derivative of thepresent invention can contain a region having affinity for the targetsubstance and a region showing the above described stimulation responseto provide an effective stimuli-responsive separating material.

[0074] The stimuli-responsive separating material of the presentinvention may be in any embodiment normally used in the art. The targetsubstance is not specifically limited. In practice, however, protein(e.g., enzyme, antibody, molecular chaperon, biological product),glycoprotein, nucleic acid, cell, artificial cell, synthetic polymer,etc. may be used.

[0075] The separating material of the present invention is a materialcontaining a region having a stimulation response utilizing the abovedescribed keto-enol tautomerization and a region having affinity for thetarget substance. The region having a stimulation response preferablycontains a substituent component represented by general formula (8).More particularly, it preferably contains a monomer componentrepresented by general formula (1) as a copolymerizable component.

[0076] The region having affinity for the target substance contains acomponent which can be bonded to or adsorbed by the target substance.More particularly, the polymer derivative of the invention preferablycontains a monomer component containing the above described componentwhich can be bonded to or adsorbed by the target substance as acomponent copolymerizable with the above described monomer componentshowing a stimulation response. The bonding of the monomer component tothe target substance does not necessarily need to be a covalent bond butmay be a bond utilizing ion complex or charge-transfer complex or bondutilizing a biochemical affinity.

[0077] Furthermore particularly, a protein such as antibody and enzyme,if any, can be bonded to the stimuli-responsive material (region havingaffinity) by making the use of the reactivity of a functional group suchas amino group and carboxyl group which is often contained in such aprotein. For example, if an amino group in a protein is used, a carboxylgroup may be incorporated in the stimuli-responsive material to producean amide bond by the following reaction formula:

Condensation Agent

R—NH₂+HOOC—R′→R—NH—CO—R′+H₂O

[0078] wherein R represents a protein; and R′ represents astimuli-responsive material

[0079] A method utilizing an aldehyde group and a method utilizing anepoxy group as mentioned below may be used:

[0080] Further, if a carboxyl group in a protein is used, an amino groupmay be incorporated in the stimuli-responsive material to produce anamide bond by the following reaction formula:

Condensation Agent

R—COOH+H₂N—R′→R—CO—NH—R′+H₂O

[0081] Further, an antibody may be incorporated in thestimuli-responsive material so that it is bonded to a protein as targetsubstance. This operation is preferably effected in phosphoric acid ortrisbuffer having a pH value in the vicinity of neutrality. The saltconcentration may be properly predetermined depending on the purpose.

[0082] Moreover, a particulate magnetic material may be bonded to thestimuli-responsive material to effect completing. In this arrangement, astimuli-responsive material to which the target substance has beenbonded or by which the target has been adsorbed can be more efficientlyagglomerated by the use of a magnet or the like during separation.

[0083] The target substance which has been bonded to or adsorbed by thestimuli-responsive material of the present invention can be easilyeluted out by any of (1) raising the salt concentration, (2) changingthe pH value (rendering the solution acidic or alkaline), (3) adding aninhibitor, substrate, etc., (4) adding a modifier such as urea and SDS,(5) adding an organic solvent, metal ion, etc. and (6) changing thetemperature.

[0084] More particularly, the stimuli-responsive separating material ofthe present invention can be applied to medicine for detecting residualagricultural chemical or diagnostic medicine and can be effectively usedfor the activation or maintenance of biological reaction by separationof biological products such as microorganisms and product of cellculture or fixing of enzyme or molecular chaperon.

[0085] In accordance with the second aspect of the present invention, asmentioned above, a thermo-responsive polymer derivative having both alower critical solution temperature (LCST) and an upper criticalsolution temperature (UCST) can be obtained by copolymerizing at leastone monomer component having a lower critical solution temperature(LCST) with at least one monomer component having an upper criticalsolution temperature (UCST).

[0086] In the second aspect of the present invention, as the monomercomponent having a lower critical solution temperature (LCST) there maybe preferably used a monomer component represented by any of the abovedescribed general formulae (2) to (5).

[0087] Particularly preferred embodiments of the various substituents ingeneral formulae (2) to (5) are described below.

[0088] R¹ preferably is a hydrogen atom or methyl group. R² and R³ eachis preferably a hydrogen atom, methyl group, ethyl group, propyl group,isopropyl group, n-butyl group, isobutyl group or tert-butyl group. R⁴is preferably a methyl group. R⁵ is preferably a methyl group, ethylgroup, propyl group, isopropyl group, n-butyl group, isobutyl group ortert-butyl group.

[0089] Specific examples of the monomer component represented by generalformula (2) include N-methyl acrylamide, N-ethylacrylamide,N-cyclopropylacrylamide, N-isopropylacrylamide, N-n-propylacrylamide,N-tert-butyl acrylamide, N-sec-butylacrylamide, N-n-butylacrylamide,N-methylmethacrylamide, N-ethylmethacrylamide, N-cyclopropylmethacrylamide, N-isopropylmethacrylamide, N-n-propyl methacrylamide,N-tert-butylmethacrylamide, N-sec-butyl methacrylamide,N-n-butylmethacrylamide, N,N-dimethyl acrylamide, N,N-diethylacrylamide,N,N-dimethyl methacrylamide, N,N-diethylmethacrylamide, N-methyl-N-ethylacrylamide, N-methyl-N-isopropylacrylamide,N-methyl-N-n-propylacrylamide, N-methyl-N-ethylmethacrylamide,N-methyl-N-isopropylmethacrylamide, isopropylmethacrylamide,N-methyl-N-n-propylmethacrylamide, N,N-diisopropylacrylamide,N,N-di-n-propylacrylamide, N,N-diisopropylmethacrylamide, and N,N-di-n-propylmethacrylamide.

[0090] Specific examples of the monomer component represented by generalformula (3) include methyl vinyl ether, and methoxy ethyl vinyl ether.

[0091] Specific examples of the monomer component represented by generalformula (4) include N-vinylacetamide, N-vinylpropionamide, N-vinylbutyrylamide, and N-vinyl isobutyrylamide.

[0092] Specific examples of the monomer component represented by generalformula (5) include N-acetylacrylamide, N-fluoroacetylacrylamide,N-propionyl acrylamide, N-butanoylacrylamide, N-pentanoylacrylamide,N-hexanoylacrylamide, N-isobutanoylacrylamide, N-benzoyl acrylamide,N-(3-fluorobenzoyl)acrylamide, N-(2, 3-difluorobenzoyl)acrylamide,N-pyridylcarbonylacrylamide, N-pyrimidylcarbonylacrylamide,N-acetylmethacrylamide, N-fluoroacetylmethacrylamide,N-propionylmethacrylamide, N-butanoylmethacrylamide,N-pentanoylmethacrylamide, N-hexanoylmethacrylamide,N-isobutanoylmethacrylamide, N-benzoylmethacrylamide,N-(3-fluorobenzoyl)methacrylamide, N-(2,3-difluorobenzoyl)methacrylamide, N-pyridylcarbonyl methacrylamide,N-pyrimidylcarbonylmethacrylamide, N-acroyl-N′-methylurea,N-acroyl-N′-ethylurea, N-acroyl-N′-fluoromethylurea,N-acroyl-N′-difluoromethylurea, N-acroyl-N′-trifluoromethylurea,N-methacroyl-N′-methylurea, N-methacroyl-N′-ethylurea,N-methacroyl-N′-fluoromethylurea, N-methacroyl-N′-difluoromethylurea,N-methacroyl-N′-trifluoromethylurea, methyl N-acroylcarbamate, ethylN-acroylcarbamate, n-butyl N-acroylcarbamate, isopropylN-acroylcarbamate, n-butyl N-acroylcarbamate, isobutylN-acroylcarbamate, t-butyl N-acroylcarbamate, fluoromethylN-acroylcarbamate, difluoromethyl N-acroylcarbamate, trifluoromethylN-acroylcarbamate, 2, 2, 2-trifluoroethyl N-acroylcarbamate, methylN-methacroylcarbamate, ethyl N-methacroylcarbamate, n-butylN-methacroylcarbamate, isopropyl N-methacroylcarbamate, n-butylN-methacroylcarbamate, isobutyl N-methacroylcarbamate, t-butylN-methacroyl carbamate, fluoromethyl N-methacroylcarbamate,difluoromethyl N-methacroylcarbamate, trifluoromethylN-methacroylcarbamate, and 2, 2, 2-trifluoroethyl N-methacroylcarbamate.

[0093] On the other hand, as the monomer component having an uppercritical solution temperature (UCST) there may be preferably used amonomer containing a substituent component represented by generalformula (8).

[0094] In the second aspect of the present invention, the compositionratio of the monomer component having a lower critical solutiontemperature (LCST) to the monomer component having an upper criticalsolution temperature (UCST) is not specifically limited but can beproperly predetermined depending on the purpose. In general, the weightratio of the monomer component having a lower critical solutiontemperature to the monomer component having an upper critical solutiontemperature is preferably from 2:1 to 1:5.

[0095] The molecular weight of the polymer derivative of the secondaspect of the present invention is not specifically limited. The polymerderivative shows little or no dependence of properties such astransition temperature on the molecular weight thereof. The molecularweight of the polymer derivative is normally from about 10² to 10⁶,preferably from about 10³ to 10⁵.

[0096] Further, in the second aspect of the present invention, thecopolymerization of a monomer component having a lower critical solutiontemperature (LCST) and a monomer component having an upper criticalsolution temperature (UCST) further with a hydrophilic or hydrophobicmonomer makes it possible to obtain thermo-responsive polymers whichexhibit a lower critical solution temperature (LCST) and an uppercritical solution temperature (UCST) within various temperature ranges.

[0097] The hydrophilic or hydrophobic monomer to be used herein is notspecifically limited. Various compounds may be used as such. Specificexamples of the hydrophilic monomer include acrylamide, allylamine,hydroxylethyl (meth)acrylate, and glycerin mono(meth)acrylate. Specificexamples of the hydrophobic monomer include ester (meth)acrylate, vinylchloride, vinylidene chloride, and styrene.

[0098] Further, the switching range (range of transition temperature) ispreferably as narrow as possible. In accordance with the second aspectof the present invention, a thermo-responsive polymer having a practicalswitching range of not higher than 10° C. can be obtained.

[0099] Further, the novel stimuli-responsive polymer derivative of thesecond aspect of the present invention is effective for the separation,fixing, calibration or control of substances which are desirably not tobe in a high temperature atmosphere. It can be effectively applied todrug delivery system (DDS), various separating agents, catheter,artificial muscle, chemovalve, etc.

[0100] A stimuli-responsive polymer derivative according to the thirdaspect of the present invention can be obtained, as mentioned above, bypolymerizing at least one monomer component represented by generalformula (6) or copolymerizing at least one monomer component representedby general formula (6) with at least one monomer component which ishydrophilic or hydrophobic with respect to the monomer component.

[0101] The monomer component represented by general formula (6) isdescribed in detail below.

[0102] Preferred examples and particularly preferred examples of R′ ingeneral formula (6) include those described with reference to R¹ ingeneral formula (1). Specific examples of the monomer represented bygeneral formula (6) include those described with reference to generalformula (1).

[0103] The hydrophilic or hydrophobic monomer to be additionallyincorporated as a copolymerizable component in the third aspect of thepresent invention cannot be unequivocally defined because it ishydrophilic or hydrophobic with respect to one monomer componentrepresented by general formula (6). Besides the monomer of generalformula (1), (meth)acrylamide and (meth)acrylic acid may be used ashydrophilic monomers and ester (meth)acrylate, vinyl chloride,vinylidene chloride, and styrene may be used as hydrophobic monomers.

[0104] In the third aspect of the present invention, as mentioned above,the incorporation of a monomer component represented by general formula(6) and optionally a monomer component which is hydrophilic orhydrophobic with respect to the monomer component represented by generalformula (6) makes it possible to obtain polymer derivatives havingvarious lower critical solution temperatures (LCST). The polymerderivative of the third aspect of the present invention loses itstransition point in an acidic or alkaline solution having apredetermined or higher acidity or alkalinity, e.g., aqueous solution ofcaustic soda having a normality of not less than 0.1 N, though dependingon the kind of the monomer component used.

[0105] The organic solvent to be added to water to develop an uppercritical solution temperature (UCST) in the third aspect of the presentinvention is not specifically limited so far as it has solubility inwater. Specific examples of the organic solvent employable hereininclude methanol, ethanol, propanol, isopropanol, acetone, THF, dioxane,acetic acid, propionic acid, ethylene glycol, and propylene glycol.

[0106] Preferred among these organic solvents are methanol, ethanol,propanol, isopropanol, acetone, and THF. These organic solvents canefficiently accelerate the agglomeration of the stimuli-responsivepolymer.

[0107] The amount of the organic solvent to be added depends on the kindof the stimuli-responsive polymer. In practice, however, it may benormally from about 5 to 50% by weight so that the lower criticalsolution temperature (LCST) disappears while a lower critical solutiontemperature (LCST) appears.

[0108] The molecular weight of the polymer derivative of the thirdaspect of the present invention is not specifically limited. The polymerderivative shows little or no dependence of properties such astransition temperature on the molecular weight thereof. In practice,however, the weight-average molecular weight of the polymer derivativeis normally from about 10² to 10⁶, preferably from about 10³ to 10⁵.

[0109] In the third aspect of the present invention, the switching rangeof the thermo-responsive polymer (range of phase transition temperature)is preferably as narrow as possible. In accordance with the third aspectof the present invention, a thermo-responsive polymer having a practicalswitching range of not higher than 10° C. can be obtained.

[0110] The novel stimuli-responsive polymer derivative of the secondaspect of the present invention is effective for the separation, fixing,calibration or control of substances which are desirably not to be in ahigh temperature atmosphere. It can be effectively applied to drugdelivery system (DDS), various separating agents, catheter, artificialmuscle, chemovalve, etc.

[0111] A thermo-responsive polymer derivative according to the fourthaspect of the present invention can be obtained, as described above, bycopolymerizing at least one monomer component represented by generalformula (6) with at least one monomer component selected from acrylamideand methacrylamide and optionally the above described hydrophilic orhydrophobic monomer component.

[0112] Preferred examples and particularly preferred examples of R¹ ingeneral formula (6) include those described with reference to R¹ ingeneral formula (1).

[0113] Specific examples of the monomer represented by general formula(6) include those described with reference to general formula (1), andN-formylacrylamide and N-formylmethacrylamide.

[0114] The hydrophilic or hydrophobic monomer to be additionallyincorporated as a copolymerizable component in the fourth aspect of thepresent invention cannot be unequivocally defined because it ishydrophilic or hydrophobic with respect to one monomer componentrepresented by general formula (6) Besides the monomer of generalformula (6), acrylamide and methacrylamide, (meth)acrylic acid, etc. maybe used as hydrophilic monomers and ester (meth)acrylate, vinylchloride, vinylidene chloride, and styrene may be used as hydrophobicmonomers.

[0115] The molecular weight of the polymer derivative of the fourthaspect of the present invention is not specifically limited. The polymerderivative shows little or no dependence of properties such astransition temperature on the molecular weight thereof. In practice,however, the weight-average molecular weight of the polymer derivativeis normally from about 10² to 10⁶, preferably from about 10³ to 10⁵.

[0116] The thermo-responsive polymer having a UCST preferably exhibitsan upper critical solution temperature of from 0 to 50° C., particularlyfrom 0 to 38° C., if it is used as a separating agent.

[0117] In the fourth aspect of the present invention, the switchingrange of the thermo-responsive polymer (range of phase transitiontemperature) is preferably as narrow as possible. In accordance with thefourth aspect of the present invention, a thermo-responsive polymerhaving a practical switching range of not higher than 10° C. can beobtained.

[0118] The novel stimuli-responsive polymer derivative of the fourthaspect of the present invention is effective for the separation, fixing,calibration or control of substances which are desirably not to be in ahigh temperature atmosphere. It can be effectively applied to drugdelivery system (DDS), various separating agents, catheter, artificialmuscle, chemovalve, etc.

[0119] In particular, the stimuli-responsive polymer derivative of thepresent invention can contain a region having affinity for the targetsubstance and a region showing the above described stimulation responseto provide an effective stimuli-responsive separating material orchemical-releasing capsule. Chemical-releasing capsules are formulationswhich release a chemical enclosed therein controllably with reversibleswelling and shrinkage due to a temperature or pH change. Theseformulations have been spotlighted as intelligent formulations which cangive a chemical in a required amount as necessary.

[0120] The stimuli-responsive separating material and chemical-releasingcapsule of the present invention may be in any embodiment normally usedin the art. The target substance is not specifically limited. Inpractice, however, protein (e.g., enzyme, antibody, molecular chaperon,biological product), glycoprotein, nucleic acid, cell, artificial cell,synthetic polymer, various chemicals (e.g., carcinostatic such asadriamycin, taxol), etc. may be used.

[0121] The separating material and chemical-releasing capsule of thepresent invention are materials having a region showing the abovedescribed stimulation response and a region having affinity for thetarget substance. The region showing a stimulation response may containa substituent component represented by general formula (6).

[0122] Further, the thermo-responsive polymer of the fourth aspect ofthe present invention, if incorporated in a chemical-releasing capsule,is preferably provided in the form of a thermo-responsive hydrogelcontaining at least one monomer component represented by general formula(6), at least one monomer component selected from acrylamide andmethacrylamide, and a crosslinking agent as a copolymerizable componentwhich is used as a chemical-releasing capsule.

[0123] As the above described crosslinking agent there is preferablyused a compound terminated by double bond at both ends thereof. Examplesof such a compound include N, N′-methylenebisacrylamide, divinylbenzene,divinylsulfone, diallyl carbinol, divinylether, and 1, 5-hexadiene.

[0124] A process for simply producing an N-acyl(meth)acrylamidederivative which can be used as a monomer of a stimuli-responsivepolymer, a process for producing an intermediate thereof and anintermediate thus produced are described below.

[0125] To date, several methods for the synthesis ofN-acyl(meth)acrylamide have been developed. However, these synthesismethods leave something to be desired in yield and productivity. Thesesynthesis methods and their problems will be described hereinafter.

[0126] The reaction of acrylamide and ketene gas as starting materialsrepresented by the following reaction formula (J. A. C. S., vol. 23, pp.915-916 (1958)) gives a good yield but requires the use of ketene gas,which is very toxic.

[0127] The reaction of acrylamide and an acid anhydride as startingmaterials represented by the following reaction formula (JP-B-37-9212(The term “JP-B” as used herein means an “examined Japanese patentpublication”)) produces Michael adducts besides N-acetylated compoundsand thus gives a poor yield.

[0128] The reaction of acrylamide and an acid chloride represented bythe following reaction formula (U.S. Pat. No. 852,460) gives muchby-products and hence a poor yield.

[0129] In order to solve the above described problems, a simpleproduction process which gives a good yield has been desired.

[0130] As a result of the extensive studies made by the presentinventors, simple production processes were found as described below.

[0131] That is, an N-acyl(meth)acrylamide derivative can be simplyproduced by reacting:

[0132] an isocyanate represented by general formula (9):

[0133] wherein R₁ represents a hydrogen atom or a methyl group; and R₂and R₃ each independently represents a hydrogen atom or a C₁₋₁₀straight-chain or branched alkyl group which may be halogenated; with

[0134] an organic metal compound represented by the following generalformula (10):

[0135] wherein M represents an alkaline metal or a halogenated alkalineearth metal; the ring A represents a cyclohexane ring, cyclopentanering, cyclopentadiene ring, pyridine ring, pyrimidine ring or benzenering; n represents a positive number of from 0 to 4; and R₄, R₅ and R₆each independently represents a hydrogen atom, a C₁₋₁₀ optionallyhalogenated straight-chain or branched alkyl group or halogen atomdirectly connected to the ring A or M, with the proviso that if n is 0,R₄ is neither a hydrogen atom nor a halogen atom, to thereby produce anN-acyl(meth)acrylamide derivative represented by general formula (11):

[0136] wherein R₁ to R₆, the ring A and n are as defined above can besimply produced.

[0137] In other words, by using an isocyanate represented by generalformula (9) instead of the conventional acrylamide as a startingmaterial and reating it with an organic metal reagent represented bygeneral formula (10), the desired N-acyl(meth)acrylamide derivative canbe simply obtained in a high yield.

[0138] In the isocyanate represented by general formula (9), R₁ to R₃preferably each represents a hydrogen atom or methyl group.

[0139] Isocyanates represented by general formula (9) and organic metalcompounds represented by general formula (10) are all commerciallyavailable or may be easily produced from commercially availablecompounds by known methods.

[0140] The solvent to be used in the above described production processis not specifically limited so far as it has no adverse effects on thereaction. Various solvents may be used so far as they do not react witha nucleophilic reagent. Such a solvent may be selected from aliphatichydrocarbon solvents such as cyclohexane, hexane and heptane, aromatichydrocarbon solvents such as benzene and toluene, halogenatedhydrocarbon solvents such as 1, 2-dichloroethane, chloroform and carbontetrachloride and ether solvents such as diethyl ether, dioxane andtetrahydrofurane (THF). These solvents may be used singly or inadmixture.

[0141] The reaction is effected normally at a temperature of from −78°C. to 70° C., preferably from −40° C. to 35° C. The reaction time is notspecifically limited. In practice, however, the reaction may beterminated when it ends in accordance with ordinary method. In general,the reaction time ranges from several minutes to 24 hours.

[0142] The compound represented by general formula (11) obtainedaccording to the present invention may be effectively used as a monomercomponent of stimuli-responsive polymer which swells or shrinks due to atemperature or pH change or an addition of a solvent or polymer such asplastic modifier optionally together with other copolymerizablecomponents. Further, analogues of this compound may be used asherbicides (see U.S. Pat. No. 852,460).

SYNTHESIS EXAMPLE 1-1

[0143] Synthesis of N-acetyl methacrylamide:

[0144] 10 ml of methacroyl isocyanate was dissolved in 50 ml of THF in aflask. To the solution was then added dropwise 35 ml of a 3 mol/l THFsolution of methyl magnesium bromide at a temperature of −20° C. in anatmosphere of nitrogen. After the termination of the dropwise addition,the mixture was then stirred at room temperature for 1 hour. To themixture were then added 100 ml of a 2 N hydrochloric acid and 100 ml ofethyl acetate sequentially. The resulting organic phase was then washedtwice with saturated brine. The solvent was then distilled off underreduced pressure. The residue thus obtained was then recrystallized fromethyl acetate to obtain 5.3 g of a colorless crystal (yield: 48%). NMRanalysis gave a strong indication that the product is the desiredcompound.

COMPARATIVE SYNTHESIS EXAMPLE 1-1

[0145] Synthesis of N-acetyl methacrylamide by conventional method:

[0146] 23.7 g of acrylamide and 60 ml of triethylamine were dissolved in100 ml of dichloromethane in a flask. To the solution was then addeddropwise 27.6 g of acetyl chloride at a temperature of −30° C. After thetermination of the dropwise addition, the mixture was then stirred at atemperature of 0° C. for 10 hours. Triethylamine hydrochloride thusprecipitated was then filtered off. The filtrate was then subjected todistillation under reduced pressure to remove the solvent therefrom. Theresidue thus obtained was then subjected to column chromatography withethyl acetate as a developing solvent and silica gel as a filler toobtain 1.5 g of the desired compound (yield: 4%).

SYNTHESIS EXAMPLE 1-2

[0147] Synthesis of N-benzoyl methacrylamide:

[0148] 2 ml of methacroyl isocyanate was dissolved in 20 ml of THF in aflask. To the solution was then added dropwise a 3 mol/l THF solution ofphenyl lithium at a temperature of −20° C. in an atmosphere of nitrogen.After the termination of the dropwise addition, the mixture was thenstirred at room temperature for 1 hour. To the mixture were then added100 ml of a 2 N hydrochloric acid and 100 ml of ethyl acetatesequentially. The resulting organic phase was then washed twice withsaturated brine. The solvent was then distilled off under reducedpressure. The residue thus obtained was then recrystallized from ethylacetate to obtain 1.3 g of a colorless crystal (yield: 40%).

[0149] NMR analysis gave a strong indication that the product is thedesired compound.

SYNTHESIS EXAMPLE 1-3

[0150] Synthesis of other N-acyl(meth)acrylamide derivatives:

[0151] The isocyanates represented by general formula (9) and theorganic metal compound represented by general formula (10) set forth inthe table below were reacted in the same manner as in Synthesis Example1-1. As a result, the desired compound (11) was obtained in a yield setforth in the table below. TABLE 1 Compound of general formula (10)Compound of Ethyl Propyl general formula Phenyl magnesium magnesium (9)lithium bromide bromide Acroyl isocyanate 55% 45% 43% Methacroyl 51% 47%44% isocyanate

[0152] In accordance with the above described production process of thepresent invention, an N-acyl(meth)acrylamide derivative which can beused as a monomer for a stimuli-responsive polymer or modifier or as astarting material of herbicide can be simply synthesized in a goodyield.

[0153] Another production process is described below.

[0154] That is, an N-acyl(meth)acrylamide derivative can be simplyproduced by reacting:

[0155] an amide represented by the following general formula (12):

[0156] wherein R₁ represents a hydrogen atom or a methyl group; and R₂and R₃ each independently represents a hydrogen atom or a C₁₋₁₀straight-chain or branched alkyl group which may be halogenated; with

[0157] a compound represented by the following general formula (13):

[0158] wherein R₄ represents a C₁₋₁₀ straight-chain or branched alkylgroup or a C₅₋₆ cyclic alkyl, aryl or heterocyclic group, each of whichmay be halogenated, to thereby produce an enamine compound representedby the following general formula (14):

[0159] wherein R₁ to R₄ are as defined above; and then allowing theenamine compound to undergo hydrolysis under acidic conditions, tothereby produce an N-acyl(meth)acrylamide derivative represented by thefollowing general formula (15):

[0160] wherein R₁ to R₄ are as defined above.

[0161] This process of the present invention is characterized by usingan acrylamide as a starting material and reacting it with a reagentrepresented by general formula (13) to produce a novel enaminerepresented by general formula (14). Further, in accordance with thisprocess of the present invention, the enamine can be hydrolyzed underacidic conditions to simply produce the desired N-acyl(meth)acrylamidein a good yield.

[0162] In the acrylamide represented by general formula (12), R₁ to R₃each preferably represent a hydrogen atom or a methyl group.

[0163] In the reagent represented by general formula (13), R₄ preferablyrepresents a methyl group, ethyl group, trifluoromethyl group,cyclohexyl group or phenyl group.

[0164] Acrylamides represented by general formula (12) and reagentsrepresented by general formula (13) are all commercially available ormay be easily produced from commercially available compounds by knownmethods.

[0165] In the synthesis of an enamine at the first stage, the reactioncan proceed without any solvent. However, some solvent is preferablyused from the standpoint of operation efficiency and yield. The solventemployable herein is not specifically limited so far as it has noadverse effects on the reaction. Various solvents may be used. Such asolvent may be selected from aliphatic hydrocarbon solvents such ascyclohexane, hexane and heptane, aromatic hydrocarbon solvents such asbenzene and toluene, halogenated hydrocarbon solvents such as 1,2-dichloroethane, chloroform and carbon tetrachloride and ether solventssuch as diethyl ether, dioxane and tetrahydrofurane (THF). Thesesolvents may be used singly or in admixture.

[0166] The first stage of the reaction is effected normally at atemperature of from 0° C. to 200° C., preferably from 40° C. to 80° C.The reaction time for the first stage is not specifically limited. Inpractice, however, the reaction may be terminated when it ends inaccordance with ordinary method. In general, the reaction time rangesfrom 30 minutes to 24 hours.

[0167] The hydrolysis reaction at the second stage can proceed withoutany solvent. However, some solvent is preferably used from thestandpoint of operation efficiency and yield. The solvent employableherein is not specifically limited so far as it has no adverse effectson the reaction. A water-soluble solvent is preferably used. Examples ofsuch a solvent include ether solvents such as dioxane andtetrahydrofurane (THF), alcohols such as methanol, ethanol, propanol andisopropanol and organic acids such as acetic acid and propionic acid.These solvents may be used singly or in admixture.

[0168] As the acidic substance to be used in the hydrolysis reactionthere may be used any acidic substance such as protonic acid, Lewis acidand organic acid without any restriction so far as it has no adverseeffects on the reaction. Examples of such an acidic substance includehydrochloric acid, sulfuric acid, nitric acid, iron chloride, copperchloride, zinc chloride, acetic acid, propionic acid and trifluoroaceticacid. These acidic substances may be used singly or in admixture.

[0169] The reaction at the second stage is effected normally at atemperature of from 0° C. to 100° C., preferably from 10° C. to 30° C.The reaction time for the second stage is not specifically limited. Inpractice, however, the reaction may be terminated when it ends inaccordance with ordinary method. In general, the reaction time rangesfrom 30 minutes to 24 hours.

[0170] The compound represented by general formula (15) obtainedaccording to the present invention may be effectively used as a monomercomponent of stimuli-responsive polymer which swells or shrinks due to atemperature or pH change or an addition of a solvent, or polymer such asplastic modifier optionally together with other copolymerizablecomponents. Further, analogues of this compound may be used asherbicides (see U.S. Pat. No. 852,460).

SYNTHESIS EXAMPLE 2-1

[0171] Synthesis of N-acetyl acrylamide:

[0172] 31 g of acrylamide and 80 g of N,N-dimethylacetamidedimethylacetal were dissolved in 200 ml of THF in a flask. The mixturewas then stirred at a temperature of 65° C. for 3 hours. Thedisappearance of the starting materials was then confirmed by gaschromatography. Thereafter, the solvent was distilled off by anevaporator. The initial distillate was distilled off under reducedpressure to obtain 40 g of (N,N-dimethylacetamide)imine in the form ofslightly yellowed liquid.

[0173] NMR analysis gave a strong indication that the product is theabove described imine substance, as follows. ¹H—NMR analysis: δ2.25(multi. 6H), δ3.10 (s. 3H), δ5.64 (multi. 1H), δ6.27 (multi. 2H) Theimine thus obtained was dissolved in a mixture of 200 ml of a 2 Nhydrochloric acid and 40 ml of acetic acid, and then stirred at roomtemperature for 4 hours. The disappearance of the imine as a startingmaterial was then confirmed by gas chromatography. Thereafter, to thesolution were added 100 ml of water and 100 ml of ethyl acetate. Theresulting organic phase was then washed with an aqueous solution ofsodium bicarbonate until it became neutral. The organic phase was thendried over magnesium sulfate. The aqueous phase was collected together,and then extracted with ethyl acetate. The resulting organic phase waswashed with an aqueous solution of sodium bicarbonate until it becameneutral, and then added to the first batch of organic phase which wasthen again dried.

[0174] The solvent was then distilled off by an evaporator. The residuewas then subjected to column chromatography with silica gel produced byMerck and ethyl acetate as a developing solvent to remove unreactedacrylamide therefrom. The fraction thus obtained was concentrated, andthen recrystallized twice from ethyl acetate to obtain 20 g of thedesired compound having a purity of 99.6% in the form of white crystal(yield: 41%).

[0175] NMR analysis gave a strong indication that the product isN-acetyl acrylamide as follows:

[0176]¹H—NMR analysis: δ2.47 (s. 3H), δ5.89 (tri. 1H), δ6.48 (d. 2H),δ7.27 (s. 1H)

COMPARATIVE SYNTHESIS EXAMPLE 2-1

[0177] Synthesis of N-acetyl acrylamide by conventional method:

[0178] 23.7 g of acrylamide and 60 ml of triethylamine were dissolved in100 ml of dichloromethane in a flask. To the solution was then addeddropwise 27.6 g of acetyl chloride at a temperature of −30° C. After thetermination of the dropwise addition, the mixture was then stirred at atemperature of 0° C. for 10 hours. Triethylamine hydrochloride thusprecipitated was then filtered off. The filtrate was then subjected todistillation under reduced pressure to remove the solvent therefrom. Theresidue thus obtained was then subjected to column chromatography withethyl acetate as a developing solvent and silica gel as a filler toobtain 1.5 g of the desired compound (yield: 4%).

SYNTHESIS EXAMPLE 2-2

[0179] Synthesis of N-acetyl methacrylamide:

[0180] 33 g of methacrylamide and 80 g of N,N-dimethylacetamidedimethylacetal were dissolved in 200 ml of THF in a flask. The mixturewas then stirred at a temperature of 65° C. for 3 hours. Thedisappearance of the starting materials was then confirmed by gaschromatography. Thereafter, the solvent was distilled off by anevaporator. The initial distillate was distilled off under reducedpressure to obtain 50 g of (N,N-dimethylacetamide)imine substance in theform of slightly yellowed liquid. NMR analysis gave a strong indicationthat the product is the desired imine.

[0181] The imine thus obtained was dissolved in a mixture of 200 ml of a2 N hydrochloric acid and 40 ml of acetic acid, and then stirred at roomtemperature for 4 hours. The disappearance of the imine as a startingmaterial was then confirmed by gas chromatography. Thereafter, to thesolution were added 100 ml of water and 100 ml of ethyl acetate. Theresulting organic phase was then washed with an aqueous solution ofsodium bicarbonate until it became neutral. The organic phase was thendried over magnesium sulfate. The aqueous phase was collected together,and then extracted with ethyl acetate. The resulting organic phase waswashed with an aqueous solution of sodium bicarbonate until it becameneutral, and then added to the first batch of organic phase which wasthen again dried.

[0182] The solvent was then distilled off by an evaporator. The residuewas then subjected to column chromatography with silica gel produced byMerck and ethyl acetate as a developing solvent to remove unreactedmethacrylamide therefrom. The fraction thus obtained was concentrated,and then recrystallized twice from ethyl acetate to obtain 30 g of thedesired compound having a purity of 99.8% in the form of white crystal(yield: 62%).

[0183] NMR analysis gave a strong indication that the product isN-acetyl methacrylamide as follows:

[0184]¹H—NMR analysis: δ2.00 (multi. 3H), δ2.50 (s. 3H), δ5.66 (qur.1H), δ5.96 (d. 1H), δ9.41 (br. s. 1H)

[0185] In accordance with the above described production process of thepresent invention, an N-acyl(meth)acrylamide derivative which can beused as a monomer for a stimuli-responsive polymer or modifier or as astarting material of herbicide can be simply synthesized via a novelenamine in a high yield.

[0186] The present invention described in greater detail with referenceto the following Examples and comparative Examples, but the presentinvention should not be construed as being limited thereto.

EXAMPLE 1-1

[0187] Synthesis of N-acetyl (meth)acrylamide (Scheme a)

[0188] In an atmosphere of nitrogen gas, 30.5 g of acrylamide, 80 g ofN,N-dimethylacetamide dimethylacetal and 400 ml of THF were charged in aflask, and then stirred at a temperature of 65° C. for 3 hours. Thereaction solution thus obtained was concentrated under reduced pressure.The residue was subjected to simple distillation under a pressure of 1mm Hg to obtain 45 g of an acroylimide. The acroylimide thus obtainedwas dissolved in 100 ml of a 2 N hydrochloric acid, and then chargedinto a flask. To the solution was then added 20 ml of acetic acid. Themixture was then stirred at room temperature for 4 hours. The reactionsolution was then extracted with ethyl acetate. The resulting organicphase was then concentrated under reduced pressure. The residue was thensubjected to column chromatography with ethyl acetate as a solvent. Theresulting fraction was then concentrated under reduced pressure. Theresidue was then recrystallized from ethyl acetate as a solvent toobtain 30 g of a white crystal.

[0189] The above described synthesis procedure was followed except that30.5 g of methacrylamide was used as a starting material. As a result,32 g of the desired compound was obtained.

[0190] NMR analysis gave a strong indication that the product is thedesired compound.

EXAMPLE 1-2

[0191] Synthesis and physical properties of poly-N-acetyl acrylamide:

[0192] In an atmosphere of nitrogen gas, 1.0 g of N-acetyl acrylamideand 10 mg of AIBN were dissolved in ethanol, and then charged into aflask where it was then stirred at a temperature of 75° C. for 3 hours.The polymer thus precipitated was withdrawn by filtration, thoroughlywashed with ethanol, and then dried at room temperature under reducedpressure to obtain 850 mg of a white solid. 50 g of the polymer thusobtained was heated and dissolved in 5 ml of a 10% ethanol solution, 20%ethanol solution and 30% ethanol solution, respectively, and thenallowed to cool. In this manner, these polymer solutions were measuredfor transparent point upon heating in the form of uniform cloudy liquid.As a result, these polymer solutions showed a transparent point of 38.5°C., 39.4° C. and 41.9° C., respectively. After reaching the transparentpoint, these polymer solutions were measured for cohesion temperatureupon cooling. As a result, these polymer solutions were observed to showUSCT at 44.7° C., 45.2° C. and 50.2° C., respectively. These polymersolutions reversibly underwent dissolution and precipitation many timesat these temperatures.

[0193] The measurement of transition temperature was effected ascalculated in terms of visible light transmittance.

[0194] The transition temperature range (temperature range requireduntil the transmittance reached from 2% to 100% upon heating or from 98%to 0% upon cooling) was as very narrow as from 2 to 6° C., thoughdepending on the ethanol concentration.

EXAMPLE 1-3

[0195] Synthesis and physical properties of poly-N-acetylmethacrylamide:

[0196] In an atmosphere of nitrogen gas, 1.0 g of N-acetylmethacrylamide and 10 mg of AIBN were dissolved in ethanol, and thencharged into a flask where it was then stirred at a temperature of 75°C. for 3 hours. The polymer thus precipitated was withdrawn byfiltration, thoroughly washed with ethanol, and then dried at roomtemperature under reduced pressure to obtain 810 mg of a white solid.

[0197] 50 mg of the polymer thus obtained was then dissolved in 5 ml ofa 1 N aqueous solution of sodium hydroxide. To the solution thusobtained was then added dropwise a 0.1 N hydrochloric acid. As a result,it was confirmed that the polymer thus obtained is a pH-responsivepolymer which repeatedly undergoes dissolution at a pH value of not lessthan 10.3 and precipitation at a pH value of not more than 10.3.

EXAMPLE 1-4

[0198] Synthesis and physical properties of N-acetyl methacrylamide andN-isopropyl acrylamide copolymer:

[0199] In an atmosphere of nitrogen gas, 1.0 g of N-acetylmethacrylamide, 1.0 g of N-isopropyl acrylamide and 10 mg of AIBN weredissolved in ethanol, and then charged into a flask where it was thenstirred at a temperature of 75° C. for 3 hours. The polymer thusprecipitated was withdrawn by filtration, thoroughly washed withethanol, and then dried at room temperature under reduced pressure toobtain 1.1 g of a white solid.

[0200] 50 mg of the polymer thus obtained was dissolved in 5 ml of abuffer having pH 1, buffer having pH 5, buffer having pH 7, bufferhaving pH 10 and buffer having pH 12, respectively. In this manner,these polymer solutions were measured for its LCST. As a result, thesepolymer solutions showed LCSTs of 52° C., 48° C., 48° C., 35° C. and 32°C., respectively. The transition temperature range (temperature rangerequired until the transmittance reached from 98% to 0%) was as verysharp as from 1.5 to 6° C., though depending on pH.

COMPARATIVE EXAMPLE 1-1

[0201] Synthesis and physical properties of poly-N-isopropyl acrylamide(PNIPAM):

[0202] In an atmosphere of nitrogen gas, 1.0 g of N-isopropyl acrylamideand 5 mg of AIBN were dissolved in ethylene glycol dimethyl ether, andthen charged into a flask where it was then stirred at a temperature of75° C. for 3 hours. The reaction solution thus obtained was thenreprecipitated from a 10/1 mixture of cyclohexane and ethyl acetate toobtain 0.6 g of a white solid.

[0203] 50 mg of the polymer thus obtained was dissolved in 5 ml of abuffer having pH 1, buffer having pH 5, buffer having pH 7, bufferhaving pH 10 and buffer having pH 12, respectively. In this manner,these polymer solutions were measured for its LCST. As a result, thesepolymer solutions were confirmed to show little or no dependence of theLCST on pH and exhibit an LCST of about 30° C.

EXAMPLE 1-5

[0204] Synthesis of trifluoroethyl N-methacroylcarbamate (Scheme b):

[0205] 4 ml of methacroyl isocyanate was dissolved in 50 ml of THF in aflask. To the solution was then added dropwise 10 ml of 2, 2,2-trifluoroethanol at a temperature of −40° C. in an atmosphere ofnitrogen. After the termination of the dropwise addition, the mixturewas then stirred at room temperature for 1 hour. The solvent was thendistilled off under reduced pressure. The residue was then subjected tosilica gel column chromatography with ethyl acetate as a developingsolvent. The fraction thus obtained was concentrated, and thenrecrystallized from ethyl acetate as a solvent to obtain 4.0 g of awhite crystal.

[0206] NMR analysis gave a strong indication that the product is thedesired compound as follows.

[0207] NMR analysis: δ2.00 (s, 3H), δ4.52 (qr, 3H), δ5.65 (s, 1H), δ5.96(s, 1H), δ8.68 (s, 1H)

EXAMPLE 1-6

[0208] Synthesis and physical properties of trifluoroethylpoly-N-methacroylcarbamate:

[0209] In an atmosphere of nitrogen gas, 1.0 g of trifluoroethylN-methacroycarbamate and 10 mg of AIBN were dissolved in ethanol, andthen charged into a flask where it was then stirred at a temperature of75° C. for 3 hours. The polymer thus precipitated was withdrawn byfiltration, thoroughly washed with ethanol, and then dried at roomtemperature under reduced pressure to obtain 520 mg of a white solid.

[0210] 50 mg of the polymer thus obtained was then dissolved in 5 ml ofa 1 N aqueous solution of sodium hydroxide. To the solution thusobtained was then added dropwise a 0.1 N hydrochloric acid. As a result,it was confirmed that the polymer thus obtained is a pH-responsivepolymer which repeatedly undergoes dissolution at a pH value of not lessthan 10.5 and precipitation at a pH value of not more than 10.5.

EXAMPLE 1-7

[0211] Synthesis of N-methacroyl-N-methylurea (Scheme c):

[0212] 15 ml of methacroyl isocyanate was dissolved in 100 ml of THF ina flask. To the solution was then added dropwise 100 ml of a 2 mol/l THFsolution of methylamine at a temperature of −40° C. in an atmosphere ofnitrogen. After the termination of the dropwise addition, the mixturewas then stirred at room temperature for 1 hour. The solvent was thendistilled off under reduced pressure. The residue was then subjected tosilica gel column chromatography with ethyl acetate as a developingsolvent. The fraction thus obtained was concentrated, and thenrecrystallized from a 10/1 mixture of ethyl acetate and ethanol as asolvent to obtain 12 g of a white crystal. NMR analysis gave a strongindication that the product is the desired compound.

EXAMPLE 1-8

[0213] Synthesis and physical properties ofpoly-N-methacroyl-N-methylurea:

[0214] In an atmosphere of nitrogen gas, 1.0 g ofN-methacroyl-N-methylurea and 10 mg of AIBN were dissolved in ethanol,and then charged into a flask where it was then stirred at a temperatureof 75° C. for 3 hours. The polymer thus precipitated was withdrawn byfiltration, thoroughly washed with ethanol, and then dried at roomtemperature under reduced pressure to obtain 880 mg of a white solid.

[0215] 50 mg of the polymer thus obtained was then dissolved in 5 ml ofa 1 N aqueous solution of sodium hydroxide. To the solution thusobtained was then added dropwise a 0.1 N hydrochloric acid. As a result,it was confirmed that the polymer thus obtained is a pH-responsivepolymer which repeatedly undergoes dissolution at a pH value of not lessthan 12.1 and precipitation at a pH value of not more than 12.1.

EXAMPLE 1-9

[0216] Preparation of immunoglobulin G-separating adsorptive material:

[0217] The separation of immunoglobulin G as target substance wasexamined using a stimuli-responsive polymer and a protein. As thestimuli-responsive polymer there was used a poly-N-acetyl acrylamide. Asthe protein there was used Protein A having a specific affinity.

[0218] 1 g of N-acryloxy succinimide and 20 g of N-acetyl acrylamidewere then subjected to polymerization with AIBN as an initiator andethylene glycol dimethyl ether as a solvent at a temperature of 70° C.for 3 hours. The solid thus precipitated was withdrawn by filtration,thoroughly washed with acetone, and then dried under reduced pressure toobtain 18 g of a copolymer.

[0219] The copolymer thus obtained and 5 g of Protein A were dissolvedin 500 ml of distilled water at a temperature of 37° C., and thenstirred for 12 hours. After the termination of the reaction, to thesolution was added 10 ml of ethanol. The temperature of the aqueoussolution was adjusted to 15° C. As a result, a copolymer containingProtein A was precipitated. The copolymer thus obtained was withdrawn byfiltration, and then thoroughly rinsed with 5° C. distilled water toobtain a stimuli-responsive separating material containing Protein A.

[0220] In an atmosphere of nitrogen, 5 g of the stimuli-responsiveseparating material thus obtained was dissolved in 1,000 ml of a 5%aqueous solution of mouse blood plasma at a temperature of 37° C., andthen stirred for 20 minutes. To the solution was then added 20 ml ofethanol. The solution was then cooled to a temperature of 10° C. tocause precipitation. The precipitate thus obtained was then rinsed withsaturated brine. The wash water was then analyzed by high-performanceliquid chromatography. As a result, it was confirmed that immunoglobulinG having a purity of 92% had been obtained.

[0221] In accordance with the first aspect of the present invention, astimuli-responsive polymer which exhibits an upper critical solutiontemperature (UCST) or a stimuli-responsive polymer which undergoesreversible dissolution and precipitation depending on the hydrogen ionconcentration or an addition of solvent can be obtained.

[0222] Further, the use of the above described stimuli-responsivepolymer makes it possible to obtain an excellent stimuli-responsiveseparating material particularly effective for the separation of atarget substance which is desirably not to be in a high temperatureatmosphere.

EXAMPLE 2-1

[0223] Synthesis and physical properties of copolymer of N-acetylacrylamide with N-isopropyl acrylamide:

[0224] 1.0 g of N-acetyl acrylamide and 200 mg of N-isopropyl acrylamidewere dissolved in 5 ml of ethanol in a three-necked flask. To thesolution was then added 5 mg of AIBN. The mixture was then stirred at atemperature of 70° C. for 4 hours. The polymer thus precipitated waswashed with ethanol, and then thoroughly dried under reduced pressure toobtain 780 mg of a copolymer (weight-average molecular weight: about7,000).

[0225] 25 mg of the copolymer thus obtained was then dissolved in 5 mlof a 15% aqueous solution of ethanol. The copolymer solution was thenmeasured for upper critical solution temperature (UCST). As a result, itwas 5° C. The same sample was then measured for lower critical solutiontemperature (LCST). As a result, it was 83° C.

[0226] The upper critical solution temperature (UCST) and the lowercritical solution temperature (LCST) were determined as calculated interms of visible light transmittance.

COMPARATIVE EXAMPLE 2-1

[0227] Synthesis and physical properties of poly-N-isopropyl acrylamide(PNIPAM):

[0228] In an atmosphere of nitrogen gas, 1.0 g of N-isopropyl acrylamideand 5 mg of AIBN were dissolved in ethylene glycol dimethyl ether, andthen charged into a flask where it was then stirred at a temperature of75° C. for 3 hours. The reaction solution thus obtained was thenreprecipitated from a 10/1 mixture of cyclohexane and ethyl acetate toobtain 0.6 g of a white solid.

[0229] 50 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was about 30° C.The aqueous solution was then allowed to stand at a temperature of 1° C.for 1 week. As a result, no polymers were observed precipitated. Thisdemonstrates that the polymer has no upper critical solution temperature(UCST).

[0230] In accordance with the second aspect of the present invention, athermo-responsive polymer having an upper critical solution temperature(UCST) and a lower critical solution temperature (LCST) can be obtained.The thermo-responsive polymer of the present invention is particularlyuseful for the separation, purification, fixing, calibration or controlof substances the working temperature of which can hardly bepredetermined (protein such as biological product, enzyme and antibody)or can be effectively used for chemovalve, etc.

EXAMPLE 3-2

[0231] Synthesis and physical properties of 1:1 copolymer of N-acetylacrylamide and N-acetyl methacrylamide:

[0232] In an atmosphere of nitrogen gas, 1.1 g of N-acetyl acrylamide,1.2 g of N-acetyl methacrylamide and 5 mg of AIBN were dissolved in 10ml of dimethyl sulfoxide, and then charged in a flask where it was thenstirred at a temperature of 75° C. for 3 hours. 200 ml of ethanol and astirrer were then put in a beaker. To ethanol was then gradually addeddropwise the above described reaction solution with vigorous stirring bya magnetic stirrer. The mixture was then stirred for 2 hours. Theresulting precipitate was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 2.0 g of a white solid. The white solid had a weight-averagemolecular weight of about 7,000.

[0233] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was 53° C.Further, the polymer solution showed a transition temperature range asvery sharp as 4° C.

[0234] Similarly, 25 mg of the same polymer was dissolved in a 0.01 Naqueous solution of caustic soda. The polymer solution was then measuredfor lower critical solution temperature (LCST). As a result, it was 66°C.

[0235] Similarly, 25 mg of the same polymer was dissolved in a 0.1 Naqueous solution of caustic soda. The polymer solution was then measuredfor lower critical solution temperature (LCST). As a result, the turbidpoint disappeared.

[0236] Similarly, 25 mg of the same polymer was dissolved in a 25%aqueous solution of ethanol. The polymer solution was then measured forlower critical solution temperature (LCST). As a result, the lowercritical solution temperature (LCST) disappeared, and an upper criticalsolution temperature (UCST) was observed at a temperature of 50° C.

[0237] The polymer reversibly underwent dissolution and precipitation atthis point many times. The transition temperature was measured ascalculated in terms of visible light transmittance.

EXAMPLE 3-3

[0238] Synthesis and physical properties of 1:4 copolymer of N-acetylacrylamide and N-acetyl methacrylamide:

[0239] In an atmosphere of nitrogen gas, 1.1 g of N-acetyl acrylamide,4.8 g of N-acetyl methacrylamide and 5 mg of AIBN were dissolved in 20ml of dimethyl sulfoxide, and then charged in a flask where it was thenstirred at a temperature of 75° C. for 3 hours. 500 ml of ethanol and astirrer were then put in a beaker. To ethanol was then gradually addeddropwise the above described reaction solution with vigorous stirring bya magnetic stirrer. The mixture was then stirred for 2 hours. Theresulting precipitate was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 5.5 g of a white solid.

[0240] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was 15° C.Further, the polymer solution showed a transition temperature range asvery sharp as 8° C.

[0241] Similarly, 25 mg of the same polymer was dissolved in a 0.1 Naqueous solution of caustic soda and a 0.01 N aqueous solution ofcaustic soda, respectively. The polymer solutions were then measured forlower critical solution temperature (LCST). As a result, the turbidpoint disappeared.

[0242] The polymer reversibly underwent dissolution and precipitation atthis point many times. The transition temperature was measured ascalculated in terms of visible light transmittance.

EXAMPLE 3-4

[0243] Synthesis and physical properties of 2:3 copolymer of N-acetylacrylamide and N-acetyl methacrylamide:

[0244] In an atmosphere of nitrogen gas, 2.2 g of N-acetyl acrylamide,2.4 g of N-acetyl methacrylamide and 5 mg of AIBN were dissolved in 20ml of dimethyl sulfoxide, and then charged in a flask where it was thenstirred at a temperature of 75° C. for 3 hours. 400 ml of ethanol and astirrer were then put in a beaker. To ethanol was then gradually addeddropwise the above described reaction solution with vigorous stirring bya magnetic stirrer. The mixture was then stirred for 2 hours. Theresulting precipitate was withdrawn by filtration, thoroughly washedwith ethanol, and then dried at room temperature under reduced pressureto obtain 4.2 g of a white solid.

[0245] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for lowercritical solution temperature (LCST). As a result, it was 23° C.Further, the polymer solution showed a transition temperature range asvery sharp as 5° C.

[0246] Similarly, 25 mg of the same polymer was dissolved in 5 ml of a0.01 N hydrochloric acid. The polymer solution was then measured forlower critical solution temperature (LCST). As a result, it was 25° C.Further, the polymer solution showed a transition temperature range asvery sharp as 4° C.

[0247] Similarly, 25 mg of the same polymer was dissolved in a 0.01 Naqueous solution of caustic soda. The polymer solution was then measuredfor lower critical solution temperature (LCST). As a result, it was 68°C.

[0248] Similarly, 25 mg of the same polymer was dissolved in a 25%aqueous solution of caustic soda. The polymer solution was then measuredfor lower critical solution temperature (LCST). As a result, the turbidpoint disappeared.

[0249] Similarly, 25 mg of the same polymer was dissolved in a 30%aqueous solution of ethanol. The polymer solution was then measured forlower critical solution temperature (LCST). As a result, the lowercritical solution temperature (LCST) disappeared, and an upper criticalsolution temperature (UCST) was observed at a temperature of 63° C.

[0250] The polymer reversibly underwent dissolution and precipitation atthis point many times. The transition temperature was measured ascalculated in terms of visible light transmittance.

COMPARATIVE EXAMPLE 3-1

[0251] Synthesis and physical properties of poly-N-isopropyl acrylamide(PNIPAM):

[0252] In an atmosphere of nitrogen gas, 1.0 g of N-isopropyl acrylamideand 5 mg of AIBN were dissolved in 5 ml of ethylene glycol dimethylether, and then charged in a flask where it was then stirred at atemperature of 75° C. for 3 hours. The resulting reaction solution was,then recrystallized from a 10/1 mixture of cyclohexane and ethyl acetateto obtain 0.6 g of a white solid.

[0253] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water, a 0.01 N hydrochloric acid, a 0.01 N aqueous solutionof caustic soda and a 0.1 N aqueous solution of caustic soda. Thepolymer solution was then measured for lower critical solutiontemperature (LCST). As a result, these polymer solutions showed littleor no dependence of the LCST on pH and a lower critical solutiontemperature (LCST) of about 30° C.

[0254] 25 mg of the polymer was dissolved in a 20% aqueous solution ofethanol. The polymer solution thus obtained was then measured for turbidpoint. As a result, the polymer solution showed a lower criticalsolution temperature (LCST) at a temperature of 19° C. but showed noupper critical solution temperature (UCST).

[0255] In accordance with the third aspect of the present invention, astimuli-responsive polymer which can switch between lower criticalsolution temperature (LCST) and upper critical solution temperature(UCST) or allow individual occurrence of reversible dissolution andprecipitation in a single compound depending on the hydrogen ionconcentration can be obtained. The stimuli-responsive polymer of thepresent invention is particularly useful for the separation,purification, fixing, calibration or control of substances the workingtemperature of which can hardly be predetermined (protein such asbiological product, enzyme and antibody) or can be effectively used forchemovalve, etc.

EXAMPLE 4-1

[0256] Synthesis and physical properties of 1:12 copolymer of N-acetylacrylamide and acrylamide:

[0257] In an atmosphere of nitrogen gas, 100 mg of N-acetyl acrylamide,1.2 g of acrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide, and then charged in a flask where it was then stirred at atemperature of 75° C. for 3 hours. 200 ml of ethanol and a stirrer werethen put in a beaker. To ethanol was then gradually added dropwise theabove described reaction solution with vigorous stirring by a magneticstirrer. The mixture was then stirred for 2 hours. The resultingprecipitate was withdrawn by filtration, thoroughly washed with ethanol,and then dried at room temperature under reduced pressure to obtain 900mg of a white solid.

[0258] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The polymer solution was then measured for uppercritical solution temperature (UCST). As a result, it was 24° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-2

[0259] Synthesis and physical properties of 1:11 copolymer of N-acetylacrylamide and acrylamide:

[0260] The procedure of polymerization reaction and purification ofExample 1 was followed except that 100 mg of N-acetyl acrylamide, 1.1 gof acrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 850 mg of awhite solid was obtained.

[0261] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 12° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-3

[0262] Synthesis and physical properties of 1:9 copolymer of N-acetylacrylamide and acrylamide:

[0263] The procedure of polymerization reaction and purification ofExample 1 was followed except that 100 mg of N-acetyl acrylamide, 900 mgof acrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 820 mg of awhite solid was obtained.

[0264] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 4° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-4

[0265] Synthesis and physical properties of 1:12 copolymer of N-acetylacrylamide and methacrylamide:

[0266] The procedure of polymerization reaction and purification ofExample 1 was followed except that 100 mg of N-acetyl acrylamide, 1.2 gof methacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 880 mg of awhite solid was obtained.

[0267] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 21° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-5

[0268] Synthesis and physical properties of 1:11 copolymer of N-acetylacrylamide and methacrylamide:

[0269] The procedure of polymerization reaction and purification ofExample 1 was followed except that 100 mg of N-acetyl acrylamide, 1.1 gof methacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 880 mg of awhite solid was obtained.

[0270] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 45° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

EXAMPLE 4-6

[0271] Synthesis and physical properties of 1:10 copolymer of N-acetylacrylamide and methacrylamide:

[0272] The procedure of polymerization reaction and purification ofExample 1 was followed except that 100 mg of N-acetyl acrylamide, 1.0 gof methacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide which was then charged in a flask. As a result, 880 mg of awhite solid was obtained.

[0273] 25 mg of the polymer thus obtained was then dissolved in 5 ml ofdistilled water. The solution thus obtained was then measured for uppercritical solution temperature (UCST). As a result, it was 55° C. Thepolymer reversibly underwent dissolution and precipitation at this pointmany times.

[0274] The measurement of the transition temperature of the samples ofExamples 1 to 6 were all effected as calculated in terms of visiblelight transmittance. Further, all these copolymers repeatedly underwentreversible dissolution and precipitation even in physiological saline,though showing slightly different upper critical solution temperatures(UCST).

EXAMPLE 4-7

[0275] Synthesis and separating properties of 1:8 copolymer of N-acetylmethacrylamide and methacrylamide having bithion fixed therein:

[0276] 100 mg of an acrylic acid ester represented by the followinggeneral formula d, 100 mg of N-acetyl acrylamide, 1.2 g ofmethacrylamide and 5 mg of AIBN were dissolved in 10 ml of dimethylsulfoxide, and then charged in a flask where it was then subjected topolymerization reaction and purification under the same conditions asmentioned above to obtain 760 mg of a white solid.

[0277] 35° C. of the polymer thus obtained was dissolved in an aqueoussolution containing 10 mg of crude avidin (purity: 85% as determined byhigh-performance liquid chromatography), and then cooled to 5° C. Theresulting precipitate was withdrawn by filtration, washed with 5° C. 10%brine, and then filtered. The resulting filtrate was dialyzed through adialysis tube, and then lyophilized to obtain 2 mg of avidin. The purityof avidin thus obtained was analyzed by high-performance liquidchromatography. As a result, it was 99.8%.

EXAMPLE 4-8

[0278] Chemical-releasing capsule:

[0279] 100 mg of N-acetyl acrylamide, 1.1 g of methacrylamide, 30 mg ofN, N′-methylene bisacrylamide and 5 mg of ammonium persulfate weredissolved in 10 ml of distilled water. The solution was then reacted ata temperature of 10° C. to prepare a gel.

[0280] The gel thus prepared was then allowed to swell in 42° C.physiological saline. To the gel was then added an aqueous solution oftaxol. In this manner, taxol was allowed to permeate into the gelovernight. Thereafter, the system was cooled to a temperature of 10° C.The gel was withdrawn, thoroughly washed with a low temperature saline,and then dipped in 38° C. physiological saline for 1 hour. The salinewas then analyzed by high-performance liquid chromatography. As aresult, taxol was confirmed released. Further, the gel was dipped in 10°C. physiological saline. The saline was then analyzed byhigh-performance liquid chromatography. The release of the chemical wassuspended. Thus, no taxol was identified.

[0281] In accordance with the fourth aspect of the present invention, athermo-responsive polymer which exhibits an upper critical solutiontemperature (UCST) in an aqueous solution, particularly in physiologicalsaline, can be obtained. The thermo-responsive polymer of the presentinvention is particularly useful for the separation, purification,fixing, calibration or control of substances the working temperature ofwhich can hardly be predetermined (protein such as biological product,enzyme and antibody) or can be effectively used for chemovalve, etc.

[0282] While the invention has been described in detail and withreference to specific examples thereof, it will be apparent to oneskilled in the art that various changes and modifications can be madetherein without departing from the spirit and scope thereof.

What is claimed is:
 1. A stimuli-responsive polymer derivative utilizingketo-enol tautomerization.
 2. The stimuli-responsive polymer derivativeaccording to claim 1, which undergoes phase transition by a change inhydrogen ion concentration or an addition of an organic solvent.
 3. Thestimuli-responsive polymer derivative according to claim 1, whichcomprises a polymerizable monomer component represented by the followinggeneral formula (1):

wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; R² represents a single bond or a C₁₋₄straight-chain or branched alkylene group which may be halogenated; R³,R⁴ and R⁵ each independently represents a hydrogen atom or a methylgroup; and X and X′ each independently represents an oxygen atom, sulfuratom, selenium atom or tellurium atom.
 4. The stimuli-responsive polymerderivative according to claim 2, which comprises a polymerizable monomercomponent represented by the following general formula (1):

wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; R² represents a single bond or a C₁₋₄straight-chain or branched alkylene group which may be halogenated; R³,R⁴ and R⁵ each independently represents a hydrogen atom or a methylgroup; and X and X′ each independently represents an oxygen atom, sulfuratom, selenium atom or tellurium atom.
 5. A thermo-responsive copolymerderivative having a lower critical solution temperature and an uppercritical solution temperature, which comprises at least one monomercomponent having a lower critical solution temperature and at least onemonomer component having an upper critical solution temperature.
 6. Thethermo-responsive copolymer derivative according to claim 5, whereinsaid monomer component having a lower critical solution temperature isat least one monomer component selected from monomers represented by thefollowing general formulae (2) to (5):

wherein R¹ represents a hydrogen atom or a methyl group; R² and R³ eachindependently represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; R⁴ represents a C₁₋₁₀ straight-chain,branched or cyclic alkyl or alkylakoxyl group which may be halogenated;R⁶ represents a C₁₋₁₀ straight-chain, branched or cyclic alkyl, alkoxyl,alkylamino, aryl or heterocyclic group which may be halogenated; and nrepresents an integer of 4 or
 5. 7. The thermo-responsive copolymerderivative according to claim 5, wherein said monomer component havingan upper critical solution temperature is at least one monomer componentrepresented by the following general formula (1):

wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; R² represents a single bond or a C₁₋₄straight-chain or branched alkylene group which may be halogenated; R³,R⁴ and R⁵ each independently represents a hydrogen atom or a methylgroup; and X and X′ each independently represents an oxygen atom, sulfuratom, selenium atom or tellurium atom.
 8. The thermo-responsivecopolymer derivative according to claim 6, wherein said monomercomponent having an upper critical solution temperature is at least onemonomer component represented by the following general formula (1):

wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; R² represents a single bond or a C₁₋₄straight-chain or branched alkylene group which may be halogenated; R³,R⁴ and R⁵ each independently represents a hydrogen atom or a methylgroup; and X and X′ each independently represents an oxygen atom, sulfuratom, selenium atom or tellurium atom.
 9. A composite stimuli-responsivepolymer derivative having a lower critical solution temperature,comprising at least one monomer component represented by the followinggeneral formula (6):

wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; and R⁵ represents a hydrogen atom or amethyl group.
 10. A thermo-responsive polymer derivative having an uppercritical solution temperature in an aqueous solution, which comprises:at least one monomer component represented by the following generalformula (6):

wherein R¹ represents a hydrogen atom or a C-₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; and R⁵ represents a hydrogen atom or amethyl group; and at least one monomer component selected fromacrylamide and methacrylamide.
 11. The thermo-responsive polymerderivative according to claim 10, wherein the charged amount of saidmonomer represented by general formula (6) is from 0.1 to 100% by weightbased on the weight of acrylamide and methacrylamide.
 12. Athermo-responsive hydrogel comprising: at least one monomer componentrepresented by the following general formula (6):

wherein R¹ represents a hydrogen atom or a C₁₋₁₀ straight-chain,branched or cyclic alkyl, alkoxyl, alkylamino, aryl or heterocyclicgroup which may be halogenated; and R⁵ represents a hydrogen atom or amethyl group; at least one monomer component selected from acrylamideand methacrylamide; and a crosslinking agent.
 13. A chemical-releasingcapsule comprising a thermo-responsive hydrogel according to claim 10.14. The responsive polymer derivative according to any one of claims 1to 11, further comprising a hydrophilic or hydrophobic monomer as acopolymerizable component.
 15. A process for producing anN-acyl(meth)acrylamide derivative, which comprises: reacting: anisocyanate represented by the following general formula (9):

wherein R₁ represents a hydrogen atom or a methyl group; and R₂ and R₃each independently represents a hydrogen atom or a C₁₋₁₀ straight-chainor branched alkyl group which may be halogenated; with an organic metalcompound represented by the following general formula (10):

wherein M represents an alkaline metal or a halogenated alkaline earthmetal; the ring A represents a cyclohexane ring, cyclopentane ring,cyclopentadiene ring, pyridine ring, pyrimidine ring or benzene ring; nrepresents a positive number of from 0 to 4; and R₄, R₅ and R₆ eachindependently represents a hydrogen atom, a C₁₋₁₀ optionally halogenatedstraight-chain or branched alkyl group or halogen atom directlyconnected to the ring A or M, with the proviso that if n is 0, R₄ isneither a hydrogen atom nor a halogen atom, to thereby produce anN-acyl(meth)acrylamide derivative represented by general formula (11):

wherein R₁ to R₆, ring A and n are the same as those defined in generalformula (9) or (10).
 16. A process for producing anN-acyl(meth)acrylamide derivative, which comprises: reacting: an amiderepresented by the following general formula (12):

wherein R₁ represents a hydrogen atom or a methyl group; and R₂ and R₃each independently represents a hydrogen atom or a C₁₋₁₀ straight-chainor branched alkyl group which may be halogenated; with a compoundrepresented by the following general formula (13):

wherein R₄ represents a C₁₋₁₀ straight-chain or branched alkyl group ora C₅₋₆ cyclic alkyl, aryl or heterocyclic group, each of which may behalogenated, to thereby produce an enamine compound represented by thefollowing general formula (14):

wherein R₁ to R₄ are the same as those defined in general formula (12)or (13); and then allowing the enamine compound to undergo hydrolysisunder an acidic condition, to thereby produce an N-acyl(meth)acrylamidederivative represented by the following general formula (15):

wherein R₁ to R₄ are the same as those defined in general formula (12)or (13).
 17. A process for producing an enamine compound, whichcomprises: reacting: an amide represented by the following generalformula (12):

wherein R₁ represents a hydrogen atom or a methyl group; and R₂ and R₃each independently represents a hydrogen atom or a C₁₋₁₀ straight-chainor branched alkyl group which may be halogenated; with a compoundrepresented by the following general formula (13):

wherein R₄ represents a C₁₋₁₀ straight-chain or branched alkyl group ora C₅₋₆ cyclic alkyl, aryl or heterocyclic group, each of which may behalogenated, to thereby produce an enamine compound represented by thefollowing general formula (14):

wherein R₁ to R₄ are the same as those defined in general formula (12)or (13).
 18. A process for producing an N-acyl(meth)acrylamidederivative, which comprises: allowing an enamine compound represented bythe following general formula (14):

wherein R₁ represents a hydrogen atom or a methyl group; R₂ and R₃ eachindependently represents a hydrogen atom or a C₁₋₁₀ straight-chain orbranched alkyl group which may be halogenated; and R₄ represents a C₁₋₁₀straight-chain or branched alkyl group or a C₅₋₆ cyclic alkyl, aryl orheterocyclic group, each of which may be halogenated, to undergohydrolysis under an acidic condition, to thereby produce anN-acyl(meth)acrylamide derivative represented by the following generalformula (15):

wherein R₁ to R₄ are the same as those defined in general formula (14).19. An enamine compound represented by the following general formula(14):

wherein R₁ represents a hydrogen atom or a methyl group; R₂ and R₃ eachindependently represents a hydrogen atom or a C₁₋₁₀ straight-chain orbranched alkyl group which may be halogenated; and R₄ represents a C₁₋₁₀straight-chain or branched alkyl group or a C₅₋₆ cyclic alkyl, aryl orheterocyclic group, each of which may be halogenated.